Cleaning of biological fluid

ABSTRACT

The present invention relates to removal of protein bound deleterious substances from an extracorporeal biological fluid by changing the affinity of the substance to the protein. The invention relates to the use of displacer substances for removal of deleterious substances. The present invention also relate to a method of removal, a system, a cleaning fluid comprising the displacer substances for removal of deleterious substances.

TECHNICAL FIELD

The present invention relates to removal of protein bound deleterioussubstances from a biological fluid by changing the affinity of thesubstance to the protein. Moreover, the invention relates to use ofC₅-C₁₀-fatty acid or salts thereof, or salicylic acid, or derivativesthereof, or salts thereof, as a displacer substance of the deleterioussubstances. The present invention also relate to a method of removal, asystem, a cleaning fluid comprising the displacer substances for removalof deleterious substances.

BACKGROUND OF THE INVENTION

The kidney function of the human body may failure for different reasons.The kidney has different functions which are crucial for survival, suchas separation and transportation of waste products from the biologicalfluid e.g. blood, balancing the level of electrolytes in the body, andbalancing the acid and base status in the body.

Failure of the kidney may be acute or chronic, and may be treated bydifferent methods like transplantation of the organ or by dialysistreatment. In dialysis treatment the biological fluids are separatedfrom the body and treated as extracorporeal fluid outside the humanbody. There are different methods of dialysis available, for examplehemodialysis, hemofiltration, and hemodiafiltration. In common, they allclean the body from waste products like urea and deleterious compoundslike uremic toxins.

Deleterious compounds, like protein bound toxins, may be present indifferent biological fluids like, for example, blood, blood plasma,peritoneal fluids. A part of the deleterious compounds bind to proteinspresent in the blood, such as albumins. The uremic toxins are in normalcases removed from the biological fluids by the kidney function, as wellas by the liver function. However, there may be sitivations where thedeleterious substances shall be removed from the biological fluid, andwhere this removal process shall take place external the human body. Forexample, the blood extracted from the body during dialysis treatment,should also be cleaned from deleterious compounds.

There are methods and means available for removal of the deleteriouscompounds. An apparatus for cleaning of extracorporeal blood is knownfrom WO 2007/046757. In this apparatus the blood is fractioned into afirst cleaned fraction and a second cleaned fraction. The second cleanedfraction is produced by removing toxins bound on proteins and/or toxinsdissolved in the plasma.

In U.S. Pat. No. 7,615,158 B2 it is described a method for removingpartially carrier bound substances from blood. The method describedtherein includes applying a pressure gradient across the membrane tocreate an ultrafiltration. A method for removal of uremic toxins boundto albumin in blood of a patient is described in U.S. Pat. No. 8,206,591B2. This method includes introducing a displacer substance into theblood such that the displacer substance displaces deleterious substancesbound to the albumin. The unbound uremic toxins are then removed byextracorporeal renal displacement treatment before the blood is returnedto the patient. The displacer substance is for example bilirubin. Thismethod is suggested to be used when drugs, such as salicylate shall beremoved from the blood.

However, there is a need to improve the possibilities to remove thedeleterious compounds from the biological fluids. Therefore, furthermeans are needed to separate and clean the extracorporeal biologicalfluids from uremic toxins.

SUMMARY OF THE INVENTION

One object of the present invention is to provide a use and a method ofremoving deleterious substances from an extracorporeal biological fluid.As the deleterious substances are substances executing a negative effecton the physiological functions of the body when present therein, it is adesire to remove these substances.

Another object of the invention is to provide a system, for removingdeleterious substances from a biological fluid by the method definedherein.

Also, one object of the invention is to provide a cleaning fluidcomprising one or more of the displacer substances described herein.

In one embodiment of the invention is a use of a substance selected fromC₅-C₁₀-fatty acid, or derivatives thereof, or salts thereof; andsalicylic acid, derivatives thereof, or salts thereof; or combinationsthereof, as displacer substance for removing a deleterious substancebound to a protein in an extracorporeal biological fluid provided.

The use of a displacer substance for removing a deleterious substancebound to a protein in an extracorporeal biological fluid, wherein thedisplacer substance is selected from C₅-C₁₀-fatty acid, or derivativesthereof, or salts thereof, salicylic acid, or derivatices thereof, orsalts thereof, or acetylsalicylic acid, or salts thereof; orcombinations thereof, is to obtain a change of affinity of thedeleterious substance to the protein.

Another embodiment is a displacer substance for use in treatment ofextracorporeal biological fluids wherein the treatment is removal ofdeleterious substances bound to proteins present in the biological fluidby using a displacer substance selected from C₅-C₁₀-fatty acid, orderivatives thereof, or salts thereof, salicylic acid, or derivativesthereof, or salts thereof, or acetylsalicylic acid, or salts thereof; orcombinations thereof.

Another embodiment is the use of displacer substance C₅-C₁₀-fatty acid,or derivatives thereof, or salts thereof. A further embodiment is theuse of displacer substance salicylic acid, derivatives thereof, or saltsthereof.

In one embodiment of the invention a use of salicylic acid andderivatives thereof, or salts thereof, as displacer substance to removea deleterious substance bound to a protein is illustrated.

An advantage with salicylic acid and derivatives, and salts thereof isits capability to bind to different sites of the albumin, with bothcompetitive and allosteric binding effect. Therefore by changing theaffinity of the deleterious substance by changing the conformity of theprotein it is possible to replace or displace the deleterious substancewith the displacer substance.

The displacer substance acts on the protein by competitive proteinbinding, or by allosteric binding, or by combination of both and by thatchanging the affinity to the protein. Thus by elaborating with theaffinity of the molecules binding to the protein it has been shown thatthe affinity of the deleterious substances may be decreased and bedisplaced of replaced by the displacer substances. This is especiallyuseful when the deleterious substance comprises an uremic toxin.

In one embodiment the deleterious substance to be removed is an uremictoxin of the group comprising the compounds p-cresol; p-cresyl sulfate;indoxyl sulfate; CMPF; and combinations thereof.

In one embodiment the protein which the deleterious substance binds tois an albumin, such as serum albumin.

In one embodiment illustrating the invention the displacer substance isa combination of salicylic acid, derivatives thereof, or salts thereofin combination with one or more C₅-C₁₀-fatty acids.

In another embodiment of the invention the displacer substance isselected from salicylic acid, derivatives thereof, or salts thereof, forexample acetyl salicylic acid.

In one embodiment of the invention a method of removing a deleterioussubstance from an extracorporeal biological fluid is provided. Thedeleterious substance binds to a protein. The method comprises thefollowing steps:

a) introducing a displacer substance into the biological fluid underconditions in which the displacer substance displaces or replacesdeleterious substance bound to the protein, thereby resulting inadditional unbound deleterious substances in the biological fluid; and

-   -   b) removing unbound deleterious substance from the biological        fluid; wherein said displacer substance is selected from        C₅-C₁₀-fatty acid, or derivatives thereof, or salts thereof; and        salicylic acid, derivatives thereof, or salts thereof; or        combinations thereof; preferably selected from salicylic acid,        or salts thereof, acetyl salicylic acid, or salts thereof, and a        C₈-fatty acid, or salts thereof, or combinations thereof, more        preferably a combination of salicylic acid and C₈-fatty acid, or        salts thereof.

The biological fluid may be any biological fluid comprising deleterioussubstances which are able to bind to a protein.The biological fluid isan extracorporeal biological fluid which is to undergo treatment byremoval of deleterious substances. Examples of biological fluids areblood, plasma, and peritoneal fluid. This list is not exhaustive.

In one embodiment the method according to step a) and step b) isperformed at temperature between 30 and 60 degrees Celsius, preferablybetween 35 and 50 degrees Celsius.

Another embodiment of the illustrated invention is a method according tothe above wherein step a) and step b) are performed at pH between 5 and8, preferably at pH between 5 and 6.

In another embodiment of the invention the removal of the deleterioussubstances according to step b) is performed by dialysis,electrodialysis, or plasmapheresis.

Also, another embodiment of the invention is a cleaning fluid comprisingone or more displacer substances. The displacer substances may beselected from C₅-C₁₀-fatty acids or salts thereof, or salicylic acid, orderivatives thereof, or salts thereof; or combinations thereof,preferably selected from salicylic acid, or salts thereof, acetylsalicylic acid, or salts thereof, and a C₈-fatty acid, or salts thereof,or combinations thereof.

A still further embodiment of the invention is a system configured toremove deleterious substances from blood. The system comprises a bloodcircuit, a fluid circuit and a blood treatment unit comprising a filtersystem including one or more semipermeable membranes separating a fluidcompartment from a blood compartment, provided with means for mixingblood and a cleaning fluid and directing the mixture obtained throughsaid blood compartment, wherein the cleaning fluid comprises displacersubstances to remove said deleterious substances bound to protein. Thedisplacer substances are selected from C₅-C₁₀-fatty acids or saltsthereof; salicylic acid, or derivatives thereof, or salts thereof; orcombinations thereof. For example, displacer substances are selectedfrom C₅-C₁₀-fatty acids or salts thereof; salicylic acid, or saltsthereof; and acetylsalicylic acid acid, or salts thereof; orcombinations thereof.

Optionally, the system is equipped with an additional unit wherein thebiological fluid, for example blood, is separated in a first fraction ofblood cells, and a second fraction of plasma. To the second fraction isa displacer substance added for removal of the deleterious substances.

The displacer substances of the invention may be selected fromC₅-C₁₀-fatty acids, or derivatives thereof, or salts thereof; orsalicylic acid, or derivatives thereof, or salts thereof; orcombinations thereof. Preferably, the displacer substance is selectedfrom sodium salicylate, sodium acetylsalicylate, and sodium octanoate(sodium salt of a C₈-fatty acid), or combinations thereof.

An additional advantage to use salicylic acid and derivatives and saltthereof as displacer substance for removing deleterious molecules frombiological fluids like blood is that they have antithrombogenic andanticoagulative properties. Otherwise, when the biological fluid isblood, an anticoagulating agent must be added separately to avoidclotting and coagulation of blood. The antithrombogenic andanticoagulative effect may be as a separate effect or as an additionaleffect to the previous.

It is well known that salicylic acid, derivatives thereof, and saltsthereof, has antioxidative and anti-inflammatory properties. Thesesubstances have also effects against diseases and syndromes like chronicinflammation, arterioscleroris and atherosclerosis.

It has surprisingly been found that salicylic acid, derivatives thereof,and salts thereof as well as C₈-C₁₀-fatty acids are useful for removalof deleterious substances from extracorporeal biological fluids.

The surprisingly findings that the displacer substances have an effecton the binding sites of albumin is an advantage for using thesesubstances in dialysis treatment and plasmapheresis. The anticoagulativeeffect is an advantage as a system where the traditional anticoagulativesubstance like heparin and citrate may be replaced by the displacersubstance as herein described.

DEFINITIONS

By the term “deleterious substances” it is herein meant substanceshaving a negative effect on the physiological functions of the body. Thedeleterious substances are for example uremic toxins.

By the term “displacer substance” it is herein meant substances whichbinds to protein, for example albumin, and the binding affinity iscompetitive, or allosteric, or both, with the deleterious substances.Thus the displacer substance may have a stronger affinity to the proteinthan the deleterious substance, and by that displace the deleterioussubstance.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIGS. 1a-e shows the result of displacing p-cresol with differentdisplacer substances. In FIG. 1a the measured amount of p-cresol whensodium salicylate was used as displacer molecule is shown. In FIG. 1 b,the measured amount of p-cresol displaced when sodium acetylsalicylatewas used as displacer substance is shown. In FIG. 1 c, the measuredamount of p-cresol displaced when sodium octanoate was used as displacersubstance. FIGS. 1d and 1e show the measured amount of p-cresoldisplaced when a combination of displacer substances was used, sodiumoctanoate and sodium salicylate, and sodium octanoate and sodiumacetylsalicylate, respectively.

FIGS. 2a-d shows the result of displacing p-cresol with addition ofdialysis solution or water, at different temperatures and pH.

DETAILED DESCRIPTION OF THE INVENTION

The human body consists of approximately 60% water, a level which isimportant to maintain for its survival. In a healthy body the level ofwater is self-regulated, by, for example, passage through the kidneys.One task of the normal kidney is to remove superfluous fluid from theblood, such as water, urea and other waste products. The resulting urineis transferred to the bladder and finally leaves the body duringurination. The kidneys second task is to regulate for example thebalance of electrolytes and acid and base in the body. Withmalfunctioning kidneys, disorders may develop in most major body organs,a syndrome called uremia. If uremia remains untreated, it will lead todeath. Uremia is treated either by kidney transplantation or some formof extracorporeal blood cleaning, e.g. hemodialysis, hemofiltration,hemodiafiltration or peritoneal dialysis.

The waste products are a large number of substances that originate fromthe body cellular metabolism. Urea is the most abundant waste productand it is produced when the proteins are metabolised. Creatinine isanother important waste product that originates from muscle metabolism.In addition to these waste products, other compounds such as proteinbound compounds and foreign substances are all excreted into the urine.

In acute kidney failure, the kidneys stop to function and to remove thewaste products, the kidney function is lost rapidly and the kidneysloose their ability to remove waste products. In most cases it is only atemporary condition, but may remain and be transferred in a chroniccondition.

Uremic toxines constitute a group of substances which are retained inpatients with kidney failure and are involved in the development andmanifestation of the uremic syndrome. These uremic retention solutes aresubdivided into three different groups according to theirphysical-chemical properties and their behaviour during dialysis therapyor treatment. Small water soluble molecules are molecules with molecularweight less than 500 Dalton (D). Examples are urea and creatinine. Thesesubstances are easily removed by dialysis. Middle (or medium) moleculesare molecules having a molecular weight range between 500 D and 15000 D,and can be removed by hemodialysis with increased efficacy withhigh-flux membrane separating.

Protein bound molecules are most often of low molecular weight but areconsidered as middle or high molecular weight molecules due to theirprotein binding. Examples are organic anions such as indoxyl sulphate(IS), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), p-cresylsulphate (PCS), p-cresol and hippuric acid (HA).

In a renal patient, the concentration of urea in water is considered tobe the same inside and outside of the cells. When urea is removedthrough dialysis from the plasma water outside the cell a concentrationdifference occurs across the cell. This, in turn, causes urea to diffuseout of the cell and thus clean the cell. The cell wall is highlypermeable to urea and the transport over the cell membrane quick.

However, other substances also to be removed from the blood are not soreadily transported over the membrane. When artificial renal procedure,like the dialysis procedures described above is required, also othermeans are needed to remove these substances from the blood. This appliesto the deleterious substances. One reason for this may be that thesubstances are mainly residing inside the cells. The substances may alsobe bound to proteins, such as albumin, residing in the plasma. Removalof these substances is therefore a challenge. Protein bound compoundsare molecules with low molecular weight, but may be considered as middleand high molecular weight molecules due to their protein binding. Theyare most often bound to albumin having a molecular weight of 66 kD.

Most of these compounds are biologically and biochemically active andhave high toxic activity, for example causing cardio-vascular damage[1,2]. Most of these compounds are organic anions, such as indoxylsulphate (IS), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF),p-cresyl sulphate (PCS), p-cresol and hippuric acid(HA). These compoundsare transported in the body by organic anion carrier molecules tovarious tissues. The compounds may be accumulated in the body, forexample in the kidneys, in tissues including endothelial cells, vascularsmooth muscle cells, osteoblasts, and the central nervous system leadingto adverse toxic effect [3]. The role of these compounds in the uremicsyndrome has been neglected for long time due to the difficulties toidentify them in the uremic serum.

A common characteristic for these compounds is their low clearance bymost of the today available dialysis strategies. For removal of thesecompounds it is only their free fraction that may be removed, thus it isrequired to remove the compound from the protein before removal. Onepossibility to remove the protein bound compound, the toxin, is tostimulate their dissociation from the binding protein, such as albumin.

One option for removal of the protein bound toxins is using thecompetitive binding which can displace the deleterious substances likeuremic toxins from their binding sites on the protein, such as albumin.Also allosteric binding is to be considered for this displacement of thedeleterious substance.

Another option is to change the pH value and/or temperature to influencethe binding between the albumin and the uremic toxins, and by thatfacilitate the dissociation of the compounds. A further option is acombination of the above, thus using the competitive or allostericbinding and changing the pH value and/or the temperature to influencethe binding between the albumin and the uremic toxins.

Human serum albumin (HSA) is the most abundant transporter or carrierprotein in plasma. The protein comprises three different domains, (I, IIand III) with each domain in turn is subdivided into two-domains, A andB. The high affinity binding sites for ligand binding are located insub-domains IIA and IIIA, also known as the Sudlow's site I and site II[4].

The albumin present in the plasma contributes to regulate the osmoticpressure and to maintain the pH level of the extravascular fluids.Furthermore, it is an important extracellular antioxidant and is acarrier protein of compounds, such as various endogenous and exogenouscompounds (such as fatty acids, hormones, and introduced molecules suchas medicinal drugs) to its two major binding sites, namely site I andsite II. Further binding sites are also present, for example suchbinding metals.

The ability of albumin to bind endogenous and exogenous compounds aswell as drugs is reduced in chronic kidney failure due to theaccumulation and the binding of the uremic toxins to this serum protein[5]. An example, the binding of bilirubin to albumin in patients withCKD is reduced due to the competitive binding of CMPF to the samebinding site, site I.

Furthermore, protein bound uremic toxins and drugs compete for the samebinding sites on the albumin molecule. Uremic toxins inhibit the bindingof many drugs to the serum albumin, for example indoxyl sulphateinhibits albumin binding of some drugs such as diazepam that binds tothe binding site (site II). CMPF is a compound which displaces drugsthat bind to binding site I of the protein, for example warfarin. Theincreased concentration of the drug may lead to an enhanced toxic effecton different organs.

The displacer substance included in the invention may be selected fromC₅-C₁₀-fatty acid, or derivatives thereof, or salts thereof; andsalicylic acid, derivatives thereof, or salts thereof; or combinationsthereof.

It is herein illustrated that C₅-C₁₀-fatty acids, or salts thereof hasan ability as displacer substance, thus having higher affinity to theprotein than the deleterious substance like for example p-cresol.

By the term “C₅-C₁₀-fatty acids” it is herein meant fatty acids with alinear or branched alkyl chain comprising C₅-C₁₀ carbon atoms. Thecarbon chain may be further substituted with substituents selected formC₁-C₃-alkyl (methyl, ethyl or propyl)-groups, -hydroxi (—OH), and —COOH.The list is not exhaustive. Examples of C₅-C₁₀-fatty acids are pentanoicacid (valeric acid), hexanoic acid (caproic acid), heptanoic acid(enanthic acid), octanoic acid (caprylic acid), nonanoic acid(pelargonic acid), and decanoic acid (capric acid). The fatty acids mayalso be included as salts thereof; an example is sodium salt of thefatty acid.

In one embodiment of the invention is the use of a C₅-C₁₀-fatty acid, orderivative thereof, or salt thereof, as a displacer substance to removea deleterious substance bound to a protein illustrated.

An example of fatty acid is octanoic acid, and its sodium salt, sodiumoctanoate, octanoic acid is also known as caprylic acid. It is to befound in human breast milk, coconut oil as well as in palm oil. Sodiumoctanoate has an anti-fungal effect and is used for the treatment andcure of fungus- and yeast infections caused by Candida albicans. It isalso used as anti-bacterial agent due to its short fatty acid chain thatcan easily penetrate membranes [6,7].

Sodium octanoate has also useful effects on patient suffering fromCrohns' disease, by inhibiting the secretion of interleukin-8 fromintestinal epithelial cells and thereby suppressing its inflammatoryeffect on the intestines [8].

Sodium octanoate can be used as a displacer substance to displacenon-covalent binding of p-cresyl sulphate, p-cresol and indoxyl sulphateto albumin Sudlow II binding site [9]. It has been reported in a studythat sodium octanoate binds with high affinity to a primary binding siteat site II. This binding leads to allosteric change in the binding siteII, which in turn results in the displacement of compounds bindingthere. It can also displace the compounds binding there in a competitiveway. The said study showed that sodium octanoate at high concentrationcan bind to albumin binding site I but with much lower binding affinity,and thereby displaces target solutes than bind to this site in acompetitive fashion [10].

Salicylic acid is another displacer substance illustrated herein, alsoas derivatives thereof, and as salt thereof. Salt of salicylic acid isfor example the sodium salicylate, included in the experiments accordingto the invention. Derivative of salicylic acid is also included in theinvention. An example of derivative is acetylsalicylic acid and saltsthereof, such as sodium acetylsalicylate. Acetylsalicylic acid is alsoknown as Aspirin® which Is a weak acid and belongs to non-steroidalanti-inflammatory drugs (NSAIDs). However, acetylsalicylic acid differsfrom the common NSAIDs by the mechanism of action. Acetylsalicylic acidis an acetyl derivative of salicylic acid. The substance is used as painrelief, as anti-inflammatory agent to suppress inflammation and as ananti-pyretic agent to reduce fever. The substance is also effective asanti-coagulation agent. The substance is also used against cancer.

Combinations of displacer substances are two or more displacersubstances selected from the groups of C₅-C₁₀-fatty acids, orderivatives thereof, or salts thereof; and salicylic acid, derivativesthereof, or salts thereof;

or combinations thereof. Examples of combinations are C₅-C₁₀-fattyacids, or derivatives thereof, or salts thereof, in combination withsalicylic acid, or salt thereof. Another example is C₅-C₁₀-fatty acids,or derivatives thereof, or salts thereof, in combination withacetylsalicylic acid, or salt thereof. Further examples are combinationof sodium octanoate and sodium salt of salicylic acid; and combinationof sodium octanoate and sodium salt of acetyl salicylic acid. Anotherexample is combination of sodium octanoate, sodium salt of salicylicacid, and sodium salt of acetyl salicylic acid.

By the term “biological fluid” it is herein meant the fluid acting ascarrier of the deleterious substances and the protein they are bound to.The biological fluid is to be cleaned from these deleterious substances,and may be, for example, blood, plasma, and peritoneal fluid. Thebiological fluid is extracorporeal biological fluid. The list ofbiological fluids is not exhaustive.

By the invention also a cleaning fluid is provided. The cleaning fluidcomprises the displacer substances as are herein described. The cleaningfluid is intended to be added in renal treatment like dialysistreatment. The cleaning fluid comprises a displacer substance selectedfrom C₅-C₁₀-fatty acid, or derivatives thereof, or salts thereof; and,salicylic acid, derivatives thereof, or salts thereof; or combinationsthereof. For example the displacer substance is C₅-C₁₀-fatty acids,derivatives thereof, or salts thereof; salicylic acid, derivativesthereof, or salts thereof; or combinations thereof. The cleaning fluiddoes also comprise physiologically acceptable excipients andelectrolytes. The cleaning fluid comprises the different components inphysiologically acceptable concentrations and has pH of between 6.5 and8.

The displacer substances for removal of deleterious molecules from abiological fluid may be included in a system, for example a system fortreatment of biological fluid such as a system for dialysis treatment ofblood. The displacer substances may be selected from C₅-C₁₀-fatty acid,or derivatives thereof, or salts thereof; and salicylic acid,derivatives thereof, or salts thereof; and or combinations thereof.

Also a system configured to remove deleterious substances from anextracorporeal biological fluid, such as blood or plasma. The system maycomprise an extracorporeal blood circuit, a fluid circuit and a bloodtreatment unit comprising a filter system including one or moresemipermeable membranes separating a fluid compartment from a bloodcompartment, provided with means for mixing blood and a cleaning fluidand directing the mixture obtained through said blood compartment,wherein the cleaning fluid comprises displacer substances to remove saiddeleterious substances bound to protein. The displacer substances areselected from C₅-C₁₀-fatty acids or salts thereof, or salicylic acid, orderivatives thereof, or salts thereof; or combinations thereof.

Optionally, the system is equipped with an additional unit wherein thebiological fluid, for example blood, is separated in a first fraction ofblood cells, and a second fraction of plasma. To the second fraction isa displacer substance added for removal of the deleterious substances.

The displacer substances could be administered as regional anticoagulantpre dialyzer, thus before the blood treatment unit in any dialysismachine. Alternatively a separate plasma separation device, such as aplasma separation filter, could be included for a separate treatment ofalbumin present in plasma with water, displacer substances, or pHadjusted water to further increase the amount of removed deleterioussubstance, e.g. uremic toxin.

An advantage of administration as regional anticoagulant should be theanti-coagulant properties from salicylic acids and the possibility todecrease the need for heparin administration. An advantage with a plasmaseparation device is that the removal of uremic toxins could befacilitated by large volumes of water and or adjustment of pH with orwithout displacer molecules.

It has been shown that removal of deleterious substance shall occur atpH of between 5 and 8, for example between 5 and 7. It has been shownthat the affinity of the deleterious substance is decreased and that theaffinity of the displacer substance is stronger within this pH range.Example of suitable pH is 5, 5.5, 6, 6.5, 7, 7.5, and 8.

The removal of the deleterious substances is also preferably performedat temperature of between 30 and 60 degree Celsius, more preferablybetween 35 and 50 degree Celsius. Examples of suitable temperature are30, 35, 36, 37, 38, 39, 40, 45, 50, 55 and 60 degree Celsius.

The displacer substances describe that release of the deleterioussubstances bound to the protein is achieved. The separation of thesesubstances from the biological fluid may be performed by techniques likedialysis, electrodialysis, plasmapheresis, etc.

When using the displacer substances that are added during dialysistreatment, the deleterious substances or uremic toxins are separatedfrom the biological fluid via the semipermeable membrane included in thesystem.

In electrodialysis the ionic substances are forced through the membraneby electric forces. In plasmapheresis, a pressure difference is createdacross the membrane so that plasma (i.e. plasma water and proteins)flows through the membrane into the second side.

The deleterious substances may also be removed from the biological fluidby a repeated addition of washing liquid. The washing liquid is forexample water or dialysis fluid, preferably water. This is especiallyapplicable on the plasma present in the blood, and also carrying thealbumin binding the deleterious substances. By adding an excessiveamount of water, some of the protein bound deleterious substances areremoved from protein and appears free in the solution. In this form itmay be easily removed by dialysis treatment of the fluid.

The conformation of the protein, the albumin, is different depending onthe pH of the surrounding liquid. The removal of deleterious substancesby water shall take place at pH 6-9, preferably at pH between 7 and 9,for example at pH 8.

The method of removing deleterious substances by repetitive addition andremoval of water comprises the following steps: a) separating of plasmafrom blood; b) adjusting the pH of the plasma, for example by adding abase, such as sodium hydroxide (NaOH) or applying a low pressure todegas carbon dioxide; c) adding of washing liquid, adjusted to pHbetween 6 and 9, for example pH 8; d) washing; e) separating the washingliquid and therein present deleterious substances which has been removedfrom the protein.

The steps c) to e) are then repeated until no or low amounts ofdeleterious substance may be detected in the washing liquid of step e).The advantages with this method is that water is cheap and readilyavailable in dialysis machines. Large amounts of water may be usedwithout excess problems of disposal. The method may be combined withhigh temperature treatment, such as a temperature between 35 and 50degrees Celsius, and by that amplify the cleaning effect.

EXAMPLES

By way of example, without any limitation of the scope, the followingexamples identify a variety of parameters pursuant to embodiments of thepresent invention.

Test of Uremic Toxins

The competitive and allosteric binding properties of displacersubstances to protein were investigated. More specifically, differentdisplacer substances capability to displace p-cresol bound to bovineserum albumin was investigated, and described in the following examples.As deleterious substance was p-cresol selected and consideredrepresentative for different uremic toxins.

Material and Methods

Centrifugation is performed in a Vivaspin® (Sartorius AG). Vivaspin® isa single tube that consists of two compartments. An upper compartmentand a lower compartment separated by a semipermeable membrane.Centrifugation is applied to force the solvent into the lowercompartment through the membrane, leaving a more concentrated sample inthe upper chamber.

-   pH measurements were performed with pH meter Orion 420 A.

Standards and Reagents:

Bovine serum albumin (BSA) (Sigma-Aldrich, purity >96%), p-cresol(deleterious substance—uremic toxin), sodium salicylate (displacersubstance and anti-coagulant, purity 99.5%), acetylsalicylic acid(displacer substance and anti-coagulant) and sodium octanoate (displacersubstance, purity 99%) were purchased from Sigma-Aldrich.

-   Methanol and Milli-Q water were used (all HPLC grade).    Sodium acetylsalicylate (the salt of acetylsalicylic acid) was    prepared by adding sodium hydroxide (NaOH) (2mol/l) to    acetylsalicylic acid (10 g) until the pH value reached 7. The    solution of acetylsalicylic acid and NaOH was frozen and the placed    in a freeze-drying machine for 16 hours to allow the frozen NaOH to    sublimate, leaving only the powder of sodium acetylsalicylate.

Analysis Method: HPLC

A 25 cm×4.6 mm Supelcosil^(TM) LC-18 column with particle size 5 μm wasused. Mobile phase A consisted of Milli-Q water and mobile phase B wasmethanol. The analytical method was isocratic with 50% of mobile phase Aand 50% mobile phase B at a flow rate of 1 ml/min. The columntemperature was 25 C and the injection volume was 20 μL. The uremicsolute (p-cresol) was quantified by using a diode array detector (DAD).The detector was monitored at wavelengths of 280 nm and 254 nm. Theoptimal UV absorption for p-cresol occurred at 280 nm. A standard curvefor p-cresol at three different concentrations (0.005 mg/ml, 0.02 mg/mland 0.04 mg/ml) was used for all p-cresol analyses.

Sample Preparation

p-cresol: Stock solutions at a concentration of 1 mg/ml were prepared bydissolving p-cresol in distilled water (Milli-Q). Working standardsolutions were prepared by appropriate dilution of the stock solutionswith Milli-Q water. Three different concentrations of p-cresol wereprepared and analysed by the HPLC to estimate the retention time andabsorbance for p-cresol:

-   Normal blood concentration of p-cresol: 0.005 mg/ml-   Uremic blood concentration of p-cresol: 0.02 mg/ml-   Maximum uremic concentration of p-cresol: 0.04 mg/ml.    Following Tests were Performed:-   Example A: Test of p-cresol binding to albumin (BSA)-   Example B: Test of p-cresol binding to albumin (BSA) in presence of    displacer substances-   Example C: Test of pH influence on the samples of Example B-   Example D: Test of influence of both temperature and pH-   Example E: Test of different temperature-   Example F: Test of p-cresol binding to albumin together with    combination of two displacer substances-   Example G: Test of p-cresol binding to albumin together with    displacer substances.

Example H: Addition of dialysis fluid to a mixture of BSA and p-cresol

-   Example I: Addition of water to a mixture BSA and p-cresol-   Example J: Test of influence of water.-   Example K: Test of influence of water at high temperature

Example A: Binding of P-Cresol to Albumin at Different pH Values

-   The ability of p-cresol, in different concentrations, to bind to the    protein (bovine serum albumin, BSA) was tested. The concentration of    BSA was kept at the level as in normal blood (40 mg/ml). The    components were mixed in water and centrifuged in a Vivaspin®    (Sartorius AG) to remove protein at 5700 rpm for 60 minutes at room    temperature.-   The samples prepared are presented in Table 1.

TABLE 1 The p-cresol concentration of samples prepared: Sample: BSA(mg/l) P-cresol (mg/ml) A1 40 0.005 A2 40 0.02 A3 40 0.04 A4 40 0.01

-   Samples were prepared by preparing samples of 1 ml containing 40    mg/ml BSA and 0.02 mg/ml p-cresol (sample A2 of table above) with pH    of 5, 7 and 8, respectively. pH was adjusted by addition of sodium    hydroxide (NaOH) or hydrochloric acid (HCl) to the solutions, as    appropriate. The pH of the samples was measured before    centrifugation and analysis by the HPLC.-   The effect of pH on the binding of p-cresol to BSA is presented in    Table 2

TABLE 2 sample A2 tested at different pH Measured concentration pH ofp-cresol (mg/ml) 5 0.0002 7 0.0032 8 0.0037

-   At pH 5 substantially no free p-cresol was detected, thus all    p-cresol was considered bound to BSA. While at pH 7 and 8 an amount    of unbound p-cresol was detected, and it was concluded that at    higher pH the binding of p-cresol to the BSA is weakened. The    weakening of the binding of p-cresol to BSA at pH 7 and 8 is    probably due to the conformational change that the protein undergoes    in pH range of 6 to 9, breakage of van der Waals interactions    between the p-cresol and BSA occurred. Thus, a conformational change    from N to B (from neutral to base) could be concluded. Further, more    p-cresol was dissociated from albumin at high pH value, especially    pH 8.

Example B: Test of P-Cresol Binding to Albumin (BSA) in Presence ofDisplacer Substances

-   Samples were prepared by dissolving BSA in water to a concentration    similar blood (40 mg/ml). p-Cresol was added to obtain an uremic    concentration (0.02 mg/ml) and displacer substances in varying    concentrations (10, 100, and 200 mg/ml).-   The displacer substances included in the examples are sodium    salicylate, sodium octanoate and sodium acetylsalicylate.-   The mixed solutions were centrifuged to remove protein at 5700 rpm    for 70 minutes at room temperature before analysis. The pH was    adjusted to 7 (physiological pH) by addition of NaOH to the samples.    The samples were injected into a HPLC. The measurement of p-cresol    by HPLC analysis was performed at room temperature.-   The samples prepared are presented in Table 3. Also the results, as    measured p-cresol concentration (mg/ml) are presented in the Table    3.

TABLE 3 Measured Sodium p-cresol p- Sodium Sodium acetyl- con- BSAcresol salicylate octanoate salicylate centration Sample (mg/ml) (mg/ml)(mg/ml) (mg/ml) (mg/ml) mg/ml B 40 0.02 0 0 0 0.0032 B1a 40 0.02 10 0 00.0048 B1b 40 0.02 100 0 0 0.0070 B1c 40 0.02 200 0 0 0.0078 B2a 40 0.020 10 0 0.0032 B2b 40 0.02 0 100 0 0.0060 B2c 40 0.02 0 200 0 0.0072 B3a40 0.02 0 0 10 0.0032 B3b 40 0.02 0 0 100 0.0043 B3c 40 0.02 0 0 2000.0067

-   Sodium salicylate has a competitive effect on the binding of    p-cresol to the protein (BSA). It can be concluded that, the higher    the concentration of sodium salisylate used in the samples, the    higher is the concentration of free p-cresol. This means that sodium    salicylate competes with p-cresol for its binding site (more    specifically site II of the protein) with the effect that p-cresol    is inhibited to bind to the albumin. Furthermore, sodium salicylate    can also bind to other sites on the albumin (e.g. site I) and induce    an allosteric changes in the region of site II, resulting in the    displacement of p-cresol binding there. Therefore, it was concluded    that sodium salicylate acts as a displacer substance and able to    displace p-cresol from its binding site on the protein.

The results of Sample B1a-B1c in Table 3 show that a fraction of freep-cresol may be detected. The concentration of unbound p-cresolincreases as the concentration of sodium salicylate is enhanced. Theconcentration of BSA was 40 mg/ml and of p-cresol was 0.02 mg/ml in allsamples. The pH was kept at 7.

Further, the results of Samples B2a-B2c show that an amount of p-cresolis unbound from the protein due to the presence of the sodium octanoatewhich displaces p-cresol from its binding site on the albumin. Theconcentration of free p-cresol is increased with the concentration ofsodium octanoate. Sodium octanoate binds with higher affinity to site IIof the protein than to site I. The p-cresol may be directly displaced oran allosteric change may be induced in the site II region and p-cresolfrom site II may occur. In Example B2c precipitation occurred.

Further, from the results of Samples B2a-B2c it may also be observedthat free p-cresol can be detected when the concentration of sodiumoctanoate is 10 mg/ml and 100 mg/ml. Also here the pH was kept at 7.

In a corresponding way, the results of Sample B3a-B3c, thus p-cresol andsodium acetylsalicylate, shows that sodium acetylsalicylate competeswith p-cresol in its binding with the protein. The higher concentrationof sodium acetylsalicylate, the more free p-cresol is detected in thesample. Sodium acetylsalicylate may also bind to other sites and induceallosteric change of site II. By the result it is confirmed that sodiumacetylsalicylate displace p-cresol from its binding site on the albumin.

The results of Samples B3a-B3c shows that free p-cresol can be detectedwhen the concentration of sodium acetylsalicylate is at least between 10and 100 mg/ml at pH 7.

Example C: Test of pH Influence on the Samples of Example B

The influence of the pH value on the binding affinity of the p-cresoland the binding competitors to the bovine serum albumin (BSA) wasinvestigated. The selected pH were pH 5, 6, 8 and 9. The pH was adjustedby adding sodium hydroxide (NaOH) or hydrochloric acid (HCl). The testswere performed with the following displacer substances: sodiumsalicylate, sodium octanoate and sodium acetylsalicylate. The resultsshow and confirm that the pH is important parameter which influences thebinding affinity of p-cresol and the displacer substances. Thedisplacement of p-cresol by the binding competitors is enhanced atspecific pH values. The effect of pH on the binding affinity agrees withthe expected effect because the albumin undergoes a conformation change(from N to B form) of the protein in the pH range 6 to 9. At pH 5 thealbumin exists in the N form. The results are present in Table 4

TABLE 4 Sample: B1a B1b B1c B2a B2b B2c B3a B3b B3c Component: Albumin(BSA) 40 40 40 40 40 40 40 40 40 p-cresol (mg/ml) 0.02 0.02 0.02 0.020.02 0.02 0.02 0.02 0.02 Sodium salicylate 10 100 200 0 0 0 (mg/ml)Sodium ctanoate 0 10 100 200 (mg/ml) Sodium acetyl 0 0 0 0 10 100 200salicylate (mg/ml) Measured p- cresol concentra- tion (×10⁻³ mg/ml) atpH: pH 5 * 7.3 14.7 17.2 8.6 — — 6.8 10.0 — pH 7 4.8 7.0 7.8 6.0 7.2 —4.3 6.7 — pH 8 2.0 0.2 0.2 3.5 4.9 8.5 0.2 3.2 — pH 9 — — — 0.2 1.0 5.8— — — * pH 6 for B2a

In Table 4 the result and the effect of pH on the binding of sodiumsalicylate and p-cresol to BSA is presented. The amount of unboundp-cresol is enhanced at low pH, especially at pH 5. Thereby, morep-cresol is displaced from its binding site on the BSA by sodiumsalicylate. This may depend on that the albumin conformation at pH 5,thus in its N-form, can bind sodium salicylate with higher affinity.Increased pH, and the binding affinity of sodium salicylate to BSAdecreases because the BSA undergoes a conformational change (from N to Bform). It is concluded that BSA in its N form binds sodium salicylatestronger than in its B form. At lower pH, pH 4, precipitation occurred.At pH 8, substantially no effect was observed, probably due to theconformation of the protein, the B-form, binds sodium salicylate withlower affinity.

Further, the result for sodium acetylsalicylate is presented. Theconcentration of free p-cresol was highest when the concentration ofsodium acetylsalicylic acid was 200 mg/ml and pH 5. Substantially nop-cresol could be detected at pH 8.

Also, the result of the study of how pH may alter the binding affinityof p-cresol and sodium octanoate to BSA is presented in Table 4 (SampleB2a-B2c). The concentration of free p-cresol was shown to increase inthe solution having low pH when sodium octanoate was present. Withincreased pH, the amount of unbound p-cresol decreases and p-cresolremained unbound to the bovine serum albumin (BSA). This effect is mostprobably depending on a conformational change of the protein in the pHrange of 6 to 9. At pH 6 the protein is in N form. It was assumed thatthe protein binds more sodium octanoate in this conformation resultingin the displacement of p-cresol form its binding sites on the albumin.At higher pH, pH values higher than 6 the albumin starts to exist morein the B form. At this pH the binding affinity is lower for sodiumoctanoate. At pH 5 precipitation of the solution was observed.Precipitation occurred in solutions with concentration of sodiumoctanoate of 100 mg/ml and 200 mg/ml at pH 6.

In Table 4 (Sample B3a-3c) corresponding result for sodiumacetylsalicylate is presented. It is shown how the binding affinitybetween p-cresol and sodium acetylsalicylicate to bovine serum albuminis altered by different pH. Solution comprising sodium acetylsalicylatewith concentration of 200 mg/ml was not included due to precipitation.

It is shown that more p-cresol is removed from its binding sites on theBSA at low pH when sodium acetylsalicylate is present. As pH increasesthe amount of unbound p-cresol is decreased. This may be due to thatalbumin in the N form has a higher binding affinity to sodium salicylateand sodium octanoate. Albumin in N-form has a higher binding affinity tosodium acetylsalicylate, which in turn can displace more p-cresol. Thebinding weakens as the pH increases, due to the conformational change tothe B-form of the protein.

Further, it is shown that the amount of unbound, free p-cresol increasesas the concentration of sodium acetylsalicylate is enhanced. Theconcentration of BSA was 40 mg/ml and the concentration of p-cresol was0.02 mg/ml in all samples tested.

Example D: Influence of Temperature and pH

-   The influence of the combination of temperature and pH on the    binding affinity of p-cresol to the bovine serum albumin (BSA) in    the presence of binding competitors was studied. The pH in the    samples was adjusted before the centrifugation at 50 ° C. (heating    cabinet).-   The tests performed regarding the influence of temperature and pH    are presented in Table 5.

TABLE 5 Sodium Sodium Sodium BSA p-cresol salicylate octanoateacetylsalicylate Example (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) D1 400.02 0 0 0 D2 40 0.02 100 0 0 D3 40 0.02 0 100 0 D4 40 0.02 0 0 100

-   The pH of each sample was adjusted in accordance with the results    obtained in Example A and Example C to maximize the effect at the    used concentration.-   The result of Example D1 to D4 is presented in Table 6.-   The measured concentration of p-cresol as a function of the    concentration of binding competitors at specific pH values was    investigated, and the effect of temperature on the binding affinity    of p-cresol and of the binding competitors to the bovine serum    albumin (BSA). The effect of temperature was clearly indicated; more    p-cresol is displaced from its binding site on the albumin by the    binding competitors at temperature of 50° C. compared with room    temperature.

TABLE 6 Test of influence of high temp and varying pH Measured p-cresolMeasured p-cresol concentration (mg/ml) concentration Example pH at roomtemperature (mg/ml) at 50° C. D1 8 0.0037 0.0065 D2 5 0.017 0.017 D3 80.0049 0.014 D4 7 0.0067 0.010

Example E: Combination of Displacer Substances at Different Temperature

-   A combination of two different binding competitors was investigated    for synergetic effect. Following compositions were tested:

TABLE 7 Sodium Sodium Sodium BSA p-cresol salicylate octanoateacetylsalicylate Composition (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) E140 0.02 10 10 0 E2 40 0.02 0 10 10

-   The compositions were tested at room temperature and at elevated    temperature of 50 ° C. for investigating the influence of    temperature on the binding affinity of p-cresol and the binding    competitors to the bovine serum albumin (BSA). pH was adjusted to pH    6 (in accordance with the results obtained in Example C.-   A combination of two binding competitors has a better effect on the    removal of p-cresol from its binding site on the albumin than a    single binding competitors acting alone on the binding site of the    protein. The pH is adjusted to 6 in all samples because as explained    in the previous mentioned results that the amount of free p-cresol    increases as the pH decreases in the presence of the binding    competitors. Furthermore, more p-cresol dissociate from the albumin    when it is heated.

TABLE 7 The measured amount of p-cresol in presence of two displacersubstances is present Measured amount of Measured amount p-cresol(mg/ml) at p-cresol (mg/ml) room temperature at 50° C. Composition E10.014 0.020 Composition E2 0.012 0.019

-   Substantially no free p-cresol was detected in the absence of    binding competitors.-   For examples (F, G, H and I) below a solution (stock solution)    having concentration of 1 mg/ml was prepared by dissolving p-cresol    (5 mg) in distilled water (Milli-Q) (5 mg). The BSA solution was    prepared from a different batch of BSA than previous preparation    (Example A). Impurities in the BSA could be concluded in the present    BSA. Different concentrations of p-cresol were prepared from the    stock solution:-   Normal blood concentration of p-cresol: 0.005 mg/ml-   Uremic blood concentration of p-cresol: 0.02 mg/ml-   Maximum uremic concentration of p-cresol: 0.04 mg/ml

Example F: Combination of Displacer Substances

-   A combination of two different binding competitors was used to    investigate the properties of the combination. Following solutions    were made:

Sodium Sodium Sodium BSA p-cresol salicylate octanoate acetylsalicylateExample (mg/ml) (mg/ml) (mg/ml) (mg/ml) (mg/ml) F1 40 0.02 10 10 0 F2 400.02 0 10 10 F1-blank 40 0 10 10 0 F2-blank 40 0 0 10 10

-   The experiments were performed at temperature of 37° C. and an    adjusted pH value of 6.

Example G: Test of Different Displacer Substances

-   Solutions of bovine serum albumin with normal blood concentration 40    mg/ml, p-cresol with the uremic concentration 0.02 mg/ml and 10    mg/ml of the binding competitors (sodium salicylate, sodium    octanoate, and sodium acetylsalicylate) were prepared. Solutions    comprising a combination of displacer substances were prepared (10    mg/ml of sodium salicylate in combination with 10 mg/ml of sodium    octanoate; 10 mg/ml of sodium octanoate in combination with 10 mg/ml    sodium acetylsalicylate). The pH of the samples was adjusted to pH    5, 5.3, 5.6, and 5.9, respectively. The amount of p-cresol displaced    in each sample was measured with HPLC as above. The result is    presented in FIGS. 1a -1 e.-   These examples confirm the experiments performed in Examples A-E    wherein another BSA was used.-   From the results presented in Example I and FIG. 1a -1 e, the same    conclusion as presented in previous examples can be drawn. It has    been shown that the binding affinity is changed by differing the pH.

Example H: Addition of Dialysis Fluid to a Mixture of BSA and P-Cresol

-   A solution containing 0.02 mg/ml p-cresol and 40 mg/ml bovine serum    albumin (BSA) were centrifuged (Vivaspin®) for 20 minutes until 0.5    ml was removed and analyzed. Then, 0.5 ml dialysis solution    (SelectBag One diluted with 37 mmol/l sodium bicarbonate and 103    mmol/l NaCl) is added to the remaining 0.5 ml solution (p-cresol and    BSA) in the centrifuge (Vivaspin) and centrifuged during 20 minutes    until 0.5 ml was removed for the analysis. This procedure was    repeated until no p-cresol could be detected by the HPLC.-   The same procedure was repeated with the solution comprising no    added p-cresol. The tests were performed at pH 8 and room    temperature.-   The results are presented in FIG. 2 a.

Example I: Addition of Water to a Mixture BSA and P-Cresol

-   1 ml of solution containing 0.02 mg/ml p-cresol and 40 mg/ml bovine    serum albumin at pH 8 was centrifuged at temperature of 37° C.    (heating cabinet) for 20 minutes until 0.5 ml was removed and    analysed. Then 0.5 ml water (Milli-Q) was added to the remaining 0.5    ml solution (p-cresol and BSA) in the centrifuge (Vivaspin®) and    centrifuged again until 0.5 ml was removed for the analysis. This    procedure was repeated 20 times, until no p-cresol could be detected    by the HPLC. The pH was adjusted to 8.-   The tests were performed at pH 8 and 37° C.-   The results are presented in FIG. 2 b.

Example J: Test of Influence of Water, Addition of Water to Mixture ofBSA and P-Cresol at pH 8

-   The influence of water addition on the binding affinity of p-cresol    to bovine serum albumin was investigated in this example, performed    at room temperature. 1 ml of a solution comprising 0.02 mg/ml    p-cresol and 40 mg/ml bovine serum albumin (BSA) was centrifuged    (Visaspin®) for 20 minutes. 0.5 ml of the solution was removed and    analysed.-   0.5 ml water (Milli-Q quality) was added to the remaining 0.5 ml    solution (p-cresol and BSA) and centrifuged (in Vivaspin®) for    another 20 minutes before another 0.5 ml was removed for the    analysis. This procedure was repeated 16 times, until no p-cresol    could be detected by the HPLC (calculated amount 0.01989 mg). This    means that almost 100% of the added p-cresol is removed from the    binding sites on BSA. The addition of water influences the binding    of peresol to BSA by breaking the van der Waals interactions between    p-cresol and BSA. Furthermore, water sets the equilibrium between    the bound and the unbound p-cresol leading to the removal of an    amount of the bound p-cresol from its binding site when a new    equilibrium is adjusted after the addition of water.-   The pH value in the stock solution (BSA and p-cresol) was chosen and    adjusted in accordance with the result in Example A to maximize the    effect.-   The results are presented in FIG. 2 c.

Example K: Addition of Water to Mixture of Protein and P-Cresol at HighTemperature (pH 8)

The previous experiment was repeated at elevated temperature. A 1 mlsolution containing 0.02 mg/ml p-cresol and 40 mg/ml bovine serumalbumin (BSA), pH 8, was centrifuged (Vivaspin®) at 50 ° C. for 20minutes until 0.5 ml was removed and analysed. Then 0.5 ml water(Milli-Q) was added to the remaining 0.5 ml solution comprising p-cresoland BSA, centrifuged for 20 minutes until 0.5 ml was removed for theanalysis. This procedure was repeated 20 times, and no p-cresol could bedetected by the HPLC. The pH was adjusted to 8 in the solution as themost effective binding affinity was observed at this pH value (ExampleA).

The p-cresol was measured after each repetition. The measured amount ofp-cresol was 0.027 mg, thus more than the added amount of p-cresol. Itwas concluded that some degradation of the sample occurred at hightemperature under action of water. It may also indicate thatcontaminations of the proteins have the same retention time as p-cresolbecause the retention time shifted towards lower value after 5 ml wasused. Furthermore, it may also be due to integration faults in thesystem. About 100% of the p-cresol is considered unbound after additionof 5 ml water and the remaining parts are contaminations.

The results are presented in FIG. 2 d.

While the invention has been described in connection with what ispresently considered to be the most practical embodiments, it is to beunderstood that the invention is not to be limited to the disclosedembodiments, but on the contrary, is intended to cover variousmodifications and equivalents included within the spirit and the scopeof the appended claims.

REFERENCES

-   [1] Barreto F. C., Barreto D. V., Liabeuf C., Meert N., Glorieux G.,    Temmar M., Choukroun G., Vanholder R., Massy Z. A., (2009), Serum    indoxyl sulphate is associated with vascular disease and mortality    in chronic kidney disease patients. Clin. J. Am. Soc. Nephrol.    4:1551-1558.-   [2] Meijers B. K., Claes K., Bammens B., de Loor H., Viaene L.,    Verbeke K., Kuypers D., Vanrenterghem Y., Evenepoel P., (2010),    p-Cresol and cardiovascular risk in mild-to-moderate kidney disease.    Clin. J. Am. Soc. Nephrol, 5:1182-1189.-   [3] Enomoto A, Niwa T., (2007), Roles of organic anion transporters    in the progression of chronic renal failure. Ther Apher Dial.    11:27-31.-   [4] Meijers B J I, Bammens B., Verbeke K., Evenepoel P., (2008) A    review of albumin binding in CKD, Am J Kidney Dis. 51:839-850.-   [5] Toshiaki S., Keishi Y., Tomoko S., Ulrich K. H., Ayaka S.,    Masaki O. (2001) Interaction Mechanism Between Indoxyl Sulfate, a    Typical Uremic Toxin Bound Site II, and Ligands Bound to Site I of    Human Serum Albumin. Pharmaceutical Research 18:520-524.-   [6] Beare-Rogers J., Dieffenbacher A., Holm J. V. (2001). Lexicon of    lipid nutrition (IUPAC technical report). Pure and applied Chemistry    73: 685-744.-   [7] www.chemspider.com/Chemical-Structure.15307.html. Retrieved on    20 Apr. 2012.-   [8] Hoshimito A., Suzuki Y., Katsuno T., Nakajima H., Saito Y.    (2002). Caprylic acid and medium-chain triglycerides inhibit IL-8    gene transcription in Caco-2 cells: comparison with the potent    histone deacetylase inhibitor trichostatin A. Br. J. Pharmacol.    136:280-6.-   [9] De Loor H., Meijers B K, Meyer T W, Bammens B., Verbeke K.,    Dehaen W., Evenepoel P. (2009) Sodium octanoate to reverse indoxyl    sulfate and p-cresyl sulfate albumin binding in uremic and normal    serum during sample preparation followed by fluorescence liquid    chromatography. J Chromatogr A. 1216: 4684-8.-   [10] Noctor T A., Wainer I W., Hage D S., (1992), Allosteric and    competetive displacement of drugs from human serum albumin by    octanoic acid, as revealed by high-performance liquid affinity    chromatography, on a human serum albumin-based stationary phase.    J.Chromatogr. 577:305-15.

1-14. (canceled)
 15. A cleaning fluid for removal of deleterioussubstances from an extracorporeal biological fluid comprising one ormore displacer substances selected from C₅-C₁₀-fatty acid, orderivatives thereof, or salts thereof, salicylic acid, or derivativesthereof, or salts thereof, or acetylsalicylic acid, or salts thereof; orcombinations thereof.
 16. (canceled)
 17. The cleaning fluid of claim 15,wherein the C₅-C₁₀-fatty acid is selected from pentanoic acid (valericacid), hexanoic acid (caproic acid), heptanoic acid (enanthic acid),octanoic acid (caprylic acid), nonanoic acid (pelargonic acid), anddecanoic acid (capric acid).
 18. The cleaning fluid of claim 15, whereinthe displacer substance comprises one or more substances selected fromCs-fatty acid, or salt thereof, salicylic acid, or salt thereof;acetylsalicylic acid, or salt thereof; or combinations thereof.
 19. Thecleaning fluid of claim 15, wherein the displacer substance comprisesone or more substances selected from Cs-fatty acid, or salts thereof,salicylic acid, or salts thereof; or combinations of Cs-fatty acid, orsalts thereof, with salicylic acid, or salts thereof, and/or withacetylsalicylic acid, or salts thereof.
 20. The cleaning fluid of claim15, wherein the displacer substance is one or more substances selectedfrom salicylic acid or sodium salt thereof, acetylsalicylic acid orsodium salt thereof, octanoic acid or sodium salt thereof; orcombination thereof.
 21. The cleaning fluid of claim 15, wherein thedisplacer substance is selected from sodium octanoate, sodiumsalicylate, and sodium acetylsalicylate, or combinations thereof. 22.The cleaning fluid of claim 15, wherein the displacer substance is acombination of sodium octanoate with one or both of sodium salicylateand sodium acetylsalicylate.
 23. The cleaning fluid of claim 15, whereinthe cleaning fluid has pH of between 6.5 and 8.